Alteration of the conserved residue tyrosine-158 to histidine renders human O-6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O-6-benzylguanine

The DNA repair protein O-6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage. O-6-Benzylguanine (BG) Is a potent inactivat or of human AGT (ED50 of 0.1 mu M) that is currently undergoing clinical tr ials to enhance chemotherapy by alkylating agents, In a screen of ACT mutan ts randomly mutated at position glycine-160, we found that the double mutan t Y158H/G160A protected Escherichea coli from killing by N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activ ity of this mutant was strongly resistant to BG (ED50 of 180 mu M). Because the single mutant G160A was not resistant to BG, this suggested that the p resence of the charged histidine residue at position 158 was responsible, T his hypothesis was confirmed by the construction of the single mutation Y15 8H. The Y158H-mutant AGT was slightly less active than wild-type AGT for th e repair of methylated DNA in vitro, but it protected E. coli from killing bgl MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 mu M. In contrast, mutant Y158F had an ED50 of 0.2 mu M. Previou s studies (M. Xu-Welliver et al,, Cancer Res., 58: 1936-1945, 1998) have sh own that mutant P140K is highly resistant to BG (ED50 of >1200 mu M). Model s of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine sid e-chain may interfere with BG binding. The double mutants P140K/Y158H and P 140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but t he use of a sensitive assay for reaction of BG with AGT indicated that thei r abilities to react were in the order P140K/Y158H < P140K < P140K/Y158F. T hese results confirm that the presence of a positively charged residue clos e to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates. Such mu tants may limit the value of BG therapy if they arise in malignant cells du ring chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoiet ic tonicity. At least 28 ACT sequences (from 25 species) have now been desc ribed, In 25 of these, the position equivalent to 158 in the human AGT is a lso a tyrosine, and in the other 3, it is a phenylalanine. The importance o f an aromatic ring side chain at this position is emphasized by previous st udies (S, Edara et al,, Carcinogenesis, 16: 1637-1642, 1995), which show th at the replacement by alanine renders human AGT inactive, Our results show that histidine can also substitute for tyrosine at this position.